Several proteins have been identified that protect Drosophila telomeres from fusion events. They include UbcD1, HP1, HOAP, the components of the Mre11-Rad50-Nbs (MRN) complex, the ATM kinase, and the putative transcription factor Woc. Of these proteins, only HOAP has been shown to localize specifically at telomeres. This study shows that the modigliani gene encodes a protein (Moi) that is enriched only at telomeres, colocalizes and physically interacts with HOAP, and is required to prevent telomeric fusions. Moi is encoded by the bicistronic CG31241 locus. This locus produces a single transcript that contains 2 ORFs that specify different essential functions. One of these ORFs encodes the 20-kDa Moi protein. The other encodes a 60-kDa protein homologous to RNA methyltransferases that is not required for telomere protection (Drosophila Tat-like). Moi and HOAP share several properties with the components of shelterin, the protein complex that protects human telomeres. HOAP and Moi are not evolutionarily conserved unlike the other proteins implicated in Drosophila telomere protection. Similarly, none of the shelterin subunits is conserved in Drosophila, while most human nonshelterin proteins have Drosophila homologues. This suggests that the HOAP-Moi complex, name in this study 'terminin,' plays a specific role in the DNA sequence-independent assembly of Drosophila telomeres. It is speculated that this complex is functionally analogous to shelterin, which binds chromosome ends in a sequence-dependent manner (Raffa, 2009).

This study has shown that the Moi protein is enriched exclusively at telomeres, where it colocalizes and physically interacts with both HOAP and HP1. Moi is not required for HOAP accumulation at telomeres, whereas Moi localization requires the wild-type functions of cav and mre11. These results suggest a mechanism for Moi localization at telomeres. It is proposed that the Drosophila chromosome ends, which contain variable DNA sequences, are processed and shaped by the MRN complex so as to allow binding of HOAP, which would in turn recruit Moi.
The Moi-HOAP complex shares several analogies with shelterin, a 6-protein complex that protects human chromosome ends, allowing cells to distinguish telomeres from sites of DNA damage (Palm, 2008). Shelterin is comprised of 3 polypeptides that directly bind the TTAGGG telomeric repeats (TRF1, TRF2, and POT1) interconnected by 3 additional proteins (Tin2, TPP1, and Rap1). The shelterin subunits share 3 properties that distinguish them from the nonshelterin telomere-associated proteins. They are specifically enriched at telomeres; they are present at telomeres throughout the cell cycle; and their functions are limited to telomere maintenance. With the exception of Tin2 and TPP1, shelterin-related proteins have been found in most eukaryotes. However, none of the shelterin subunits are conserved in Drosophila. This is not surprising as Drosophila telomeres are DNA sequence-independent structures, while the core subunits of shelterin are sequence-specific DNA binding proteins (Raffa, 2009 and references therein).

The Moi and HOAP proteins have the same properties of the shelterin subunits: they accumulate only at telomeres; they are likely to be associated with telomeres throughout the cell cycle, as they colocalize in discrete aggregates present in all interphase nuclei and are enriched at polytene chromosome telomeres; and they appear to function only at telomeres. HP1 interacts with both Moi and HOAP but does not share their properties; it localizes to multiple chromosomal sites and its function is not limited to telomere maintenance. Notably, Moi and HOAP are not conserved in either yeasts or mammals, consistent with the fact that both proteins associate with telomeres in a sequence-independent fashion. Thus, it is proposed that Moi and HOAP are the founding components of a Drosophila telomere complex, named here 'terminin,' which acts like human shelterin. It is suggested that terminin accumulation at chromosome ends prevents both checkpoint activation and telomere fusion and helps in recruiting nonterminin components of Drosophila telomeres. This hypothesis posits that the nonterminin proteins of Drosophila telomeres should be conserved in humans and play roles in telomere maintenance. Similarly, nonshelterin components of human telomeres should have conserved Drosophila homologues. Indeed, all of the nonterminin proteins specified by the Drosophila telomere-fusion mutants so far identified have human counterparts. UbcD1 and Woc have highly conserved human homologues but it is currently unknown whether any of them is involved in telomere maintenance. HP1 too is conserved in humans, and HP1 homologues have been found at mouse telomeres where they appear to control telomere length. The ATM kinase and the components of the MRN complex (Mre11, Rad50, and Nbs) have highly conserved human orthologues (Zhu, 2000; Karlseder, 2004), which bind shelterin and help to regulate human telomere organization (Raffa, 2009).

In addition to ATM, Mre11, Rad50, and Nbs1, the human nonshelterin factors include Ku70 and Ku80 and their associated DNA-dependent protein kinase catalytic subunit (DNA-PKcs), the ATR kinase, PARP1 and PARP2, Rad51, the ERCC1/XPF endonuclease, the Apollo nuclease, and the RecQ family members WRN and BLM, which are mutated in the Werner and Bloom syndrome, respectively (Palm, 2008). With the exception of DNA-PKcs, all these nonshelterin proteins have Drosophila homologues. There is also evidence that Drosophila ATM and ATR cooperate to prevent telomere fusion, and that Ku70 and Ku80 act as negative regulators of Drosophila telomere elongation by transposition. However, it is not currently known whether the fly homologues of Rad51, ERCC1, Apollo, WRN, and BLM play roles at Drosophila telomeres (Raffa, 2009).

In summary, it is clear that the terminin and shelterin components are not evolutionarily conserved. In contrast, the nonterminin and nonshelterin proteins are largely conserved from flies to mammals, and many of them play telomere-related functions in both Drosophila and humans. This suggests that the main difference between Drosophila and human telomeres is in the protective complexes that specifically associate with the DNA termini. Thus, apart from the different mechanisms of elongation, Drosophila and human telomeres might not be as different as it is generally thought (Raffa, 2009).

A product of the bicistronic Drosophila melanogaster gene CG31241, which also encodes a trimethylguanosine synthase, plays a role in telomere protection

Although telomere formation occurs through a different mechanism in Drosophila compared with other organisms, telomere associations result from mutations in homologous genes, indicating the involvement of similar pathways in chromosome end protection. This study reports that mutations of the Drosophila gene CG31241 lead to high frequency chromosome end fusions. CG31241 is a bicistronic gene that encodes trimethylguanosine synthase (TGS1), which forms the m3G caps of noncoding small RNAs, and a novel protein, DTL (Modigliani). Although TGS1 has no role in telomere protection, DTL is localized at specific sites, including the ends of polytene chromosomes, and its loss results in telomere associations. Mutations of ATM- and Rad3-related (ATR) kinase suppress telomere fusions in the absence of DTL. Thus, genetic interactions place DTL in an ATR-related pathway in telomere protection. In contrast to ATR kinase, mutations of ATM (ataxia telangiectasia mutated) kinase, which acts in a partially overlapping pathway of telomere protection, do not suppress formation of telomere associations in the absence of DTL. Thus, uncovering the role of DTL will help to dissect the evolutionary conserved pathway(s) controlling ATM-ATR-related telomere protection (Komonyi, 2009).

In agreement with observations that the presence of specific sequences at chromosome ends does not ensure telomere protection in Drosophila, this study demonstrated the presence of telomere-specific HeT-A transposon sequences at CG31241 mutant chromosome ends by in situ hybridization (Komonyi, 2009).

Independently of telomeric DNA sequences, nucleoprotein complexes containing the telomere-specific protein HOAP can assemble at Drosophila chromosome ends (Cenci, 2003). The gene encoding HOAP was identified by caravaggio (cav) mutations and it was found that approximately 99% of cav mutant metaphases contained chromosome end-to-end attachments (Cenci, 2003). It was the therefore asked whether the DTL protein had a role in localizing the telomere-specific HOAP protein to chromosome ends. By immunostaining, the presence of HOAP was detected both at polytene and mitotic chromosome ends of CG31241 mutants. Furthermore, HOAP-specific staining could be detected at mitotic chromosome fusions in CG31241 mutant neuroblasts. Similarly, the localization of another telomere-associated factor HP1, whose absence also results in telomere associations, could be detected at least at polytene CG31241 chromosome ends. A complete loss of either HOAP or HP1 from chromosome ends could not thus explain the telomere associations observed in CG31241 mutant cells. To determine whether the DTL protein is also localized specifically to chromosome ends, a Flag-labeled DTL was ectopically expressed from a transgene using an Act5 promoter. The expression of this epitope-tagged protein in CG31241 mutants, which also express a TGS1 transgene, rescued all observable dtl phenotype studied, and resulted in the development of fully-fledged adult. DTL localization was observed at selected interband regions on each chromosome arm and specific staining was also detectable at chromosome ends. A Flag-specific signal was detected only in transgene carriers and its localization was reproducible both at specific interbands and chromosome ends. Interestingly, the general distribution of DTL-Flag-specific signal was different to that obtained with either HOAP- or HP1-specific antibodies, and at some chromosome ends DTL-Flag did not seem to be localized at the very end of the polytene chromosome. However, the fact that the chromosomal distribution of DTL was detected in animals ectopically expressing the protein should be taken into account in the interpretation of this result (Komonyi, 2009).

In chromosome end protection, ATM acts in a common pathway with members of the MRN (Mre11-Rad50-Nbs1) complex and mutations affecting ATM or MRN subunits result in a phenotype showing strong telomere association. By contrast, mutations affecting ATR (mei-41/atr) and the gene encoding its associated factor ATRIP (mus304) do not induce telomere associations. A role of ATR in end protection, however, was revealed from observations that mei-41/atr; tefu/atm double mutants, as well as other combinations of mutants of the ATM- and ATR-associated pathways displayed significantly stronger telomere association phenotypes than tefu/atm mutants alone. CG31241 mutations, in combination with a tefu/atm-null mutation, caused lethality in an earlier phase than either mutation alone, and the number of anaphase chromosome bridges in neuroblasts of tefu/atm CG31241 double mutants was as high or even higher than either mutation alone. Both the viability and telomere association phenotype of double mutants depend on the strength of the CG31241 allele used. Thus, the genetic interaction indicates that DTL has a role in a telomere protection pathway other than the one in which ATM acts. DTL does not seem to have a role in ATM checkpoint control function, because, in contrast to tefu/atm mutants, CG31241 mutants displayed mitotic arrest following low-dose X-ray irradiation (Komonyi, 2009).

The molecular mechanism by which DTL participates in telomere maintenance is unknown. The unusual organization of the CG31241 gene in that it also encodes TGS1, raises the question of whether DTL has a role in telomere maintenance through RNA modification. Although a role for TMG-cap-containing RNAs in both yeast and human telomere formation has been suggested, there is no data indicating that a specific RNA is involved in chromosome end protection in Drosophila. The current data do not suggest a role for TMG-cap-containing RNA(s) in Drosophila telomeres, and CG31241 alleles that lose TGS1 function but retain DTL function do not display telomere associations (Komonyi, 2009).

DTL seems to be a Drosophila-specific protein. The putative products of CG31241-related genes of different Drosophila species show strong conservation in both the DTL and TGS1 ORFs. Although the putative DTL proteins have conserved amino acid blocks, they contain no domains or motifs identified in databases, except weak similarity to a region characteristic of prokaryotic helicases. Several putative phosphorylation sites were identified among the conserved amino acids of DTL proteins, but whether these have specific roles remains to be determined. Nonetheless, because the involvement of ATM- and ATR-related pathways in telomere protection seem to be conserved across the entire kingdom of eukaryotes, it is likely that flies also use these conserved pathways despite their different mechanism of telomere formation. Therefore, it is believed that the identification of DTL and its interaction with ATR will facilitate studies to uncover the existence of similar functions in other organisms, which, in turn, could lead to novel insights into carcinogenesis (Komonyi, 2009).

Verrocchio, a Drosophila OB fold-containing protein, is a component of the terminin telomere-capping complex

Drosophila telomeres are elongated by transposition of specialized retroelements rather than telomerase activity, and are assembled independently of the terminal DNA sequence. Drosophila telomeres are protected by terminin, a complex that includes the HOAP (Heterochromatin Protein 1/origin recognition complex-associated protein) and Moi (Modigliani) proteins and shares the properties of human shelterin. This study shows that Verrocchio (Ver), an oligonucleotide/oligosaccharide-binding (OB) fold-containing protein related to Rpa2/Stn1, interacts physically with HOAP and Moi, is enriched only at telomeres, and prevents telomere fusion. These results indicate that Ver is a new terminin component; it is speculated that, concomitant with telomerase loss, Drosophila evolved terminin to bind chromosome ends independently of the DNA sequence (Raffa, 2010).

It has been suggested that fly telomeres are capped by the HOAP-Moi complex, which was called terminin, and which has the same properties of shelterin: a specific telomeric localization throughout the cell cycle, and a telomere-limited function
(Raffa, 2009). This study has shown that ver mutants exhibit a very high frequency of telomeric fusions (about five per cell), comparable with those observed previously in cav (HOAP) and moi mutants (Cenci, 2003; Musarò, 2008; Raffa, 2009). Consistent with these findings, Ver is enriched exclusively at telomeres like HOAP and Moi, and colocalizes precisely and interacts physically with both these proteins. In addition, the current analyses indicate that Ver functions only at telomeres. These findings strongly suggest that Ver is a component of the terminin complex (Raffa, 2010).

The results indicate that Ver contains an OB fold domain that shares structural similarity with the Rpa2/Stn1 OB fold. Interestingly, the Drosophila genome does not appear to contain homologs of the shelterin subunits and the other CST subunits. However, all of the nonshelterin and non-CST components of human telomeres are conserved in flies. Conversely, with the exception of HOAP and Moi, all of the Drosophila telomere-related proteins identified so far have clear human counterparts (Cenci, 2005; Raffa, 2009). Thus, it is hypothesized that, concomitant with telomerase loss, Drosophila lost the shelterin and the CST homologs that bind DNA in a sequence-specific fashion, and evolved terminin to bind chromosome ends independently of the DNA sequence (Raffa, 2010).

The hypothesis on terminin evolution generates several expectations. It is logical to assume that telomerase loss resulted in a divergence of terminal DNA sequences, accompanied by a strong selective pressure toward the evolution of sequence-independent telomere-binding factors. It is also conceivable that the evolutionary pressure on these factors was higher than that exerted on telomere proteins not specifically involved in capping. Therefore, one would predict that proteins involved directly and exclusively in telomere capping evolved more rapidly than the other telomere-associated proteins. This prediction is verified by the finding that HOAP, Moi, and Ver are fast-evolving proteins, while the other Drosophila telomere proteins, including HP1, are not (Raffa, 2010).

Although the frequencies of telomeric fusions elicited by loss of each terminin component are fully comparable, Ver, Moi, and HOAP do not play identical roles at Drosophila telomeres. HOAP localizes at telomeres independently of Ver and Moi, which are both HOAP-dependent and mutually dependent for telomeric localization. In addition, while loss of HOAP triggers both the DNA damage and the spindle assembly (SAC) response (Musarò, 2008), depletion of either Ver or Moi (Raffa, 2009) does not appear to elicit these checkpoint responses. These results suggest that HOAP is crucial for masking chromosome ends to avoid their recognition as double-strand breaks. Ver and Moi are not required for terminal DNA protection so as to prevent checkpoint responses. However, Ver and Moi are essential to hide chromosome ends from the DNA repair machineries that mediate telomere fusion. A Ver protein with mutations in the OB fold domain is still recruited at telomeres, but is unable to prevent telomere fusion. This suggests that the integrity of the Ver OB fold domain is crucial to prevent inappropriate repair of terminal DNA, and implies that Drosophila telomeres terminate with a single-strand overhang like their yeast, plant, and mammalian counterparts (Raffa, 2010).

Drosophila telomeres are sequence-independent structures maintained by transposition to chromosome ends of three specialized retroelements rather than by telomerase activity. Fly telomeres are protected by the terminin complex that includes the HOAP, HipHop, Moi and Ver proteins. These are fast evolving, non-conserved proteins that localize and function exclusively at telomeres, protecting them from fusion events. It has been suggested that terminin is the functional analogue of shelterin, the multi-protein complex that protects human telomeres. This study used electrophoretic mobility shift assay (EMSA) and atomic force microscopy (AFM) to show that Ver preferentially binds single-stranded DNA (ssDNA) with no sequence specificity. It was also shown that Moi and Ver form a complex in vivo. Although these two proteins are mutually dependent for their localization at telomeres, Moi neither binds ssDNA nor facilitates Ver binding to ssDNA. Consistent with these results, Ver-depleted telomeres were found to form RPA and γH2AX foci, like the human telomeres lacking the ssDNA-binding POT1 protein. Collectively, these findings suggest that Drosophila telomeres possess a ssDNA overhang like the other eukaryotes, and that the terminin complex is architecturally and functionally similar to shelterin (Cicconi, 2016).

Dealing with chromosome ends represents a major problem for the cell, as they can be mistaken for double strand breaks (DSBs) and activate the DNA damage response (DDR), leading to unwanted repair, telomere fusion and genome instability. Different organisms evolved different protein complexes that specifically bind chromosome ends and help assembly of the telomere, a protective structure that shields DNA termini preventing DSB signaling. In most eukaryotes, telomeric DNA consists of short tandem repeats added by telomerase to chromosome ends. Replication of the lagging strand results in the formation of a terminal 3' G-rich overhang; completion of telomere replication through a fine interplay between exonuclease activities and fill-in DNA synthesis results in 3' overhangs of appropriate length at the ends of both sister chromatids (Cicconi, 2016).

In organisms with telomerase, terminal repeats are specifically recognized by specialized telomere capping complexes. In humans, the TTAGGG repeats are selectively bound by the six-protein (TRF1, TRF2, Rap1, TIN2, TPP1, POT1) shelterin complex, which localizes and function almost exclusively at telomeres. TRF1 and TRF2 bind the TTAGGG duplex and POT1 the 3' overhang; TIN2 and TPP1 bridge POT1 to TRF1 and TRF2. hRap1, a distant homologue of Saccharomyces cerevisiae Rap1, interacts with TRF2, but is not directly implicated in telomere protection or length regulation. TRF2 dysfunction triggers the ATM signaling pathway, and leads to the accumulation of telomere dysfunction foci (TIFs) enriched in γ-H2AX. Loss of POT1 causes the accumulation of RPA (Replication protein A) onto the 3' overhang, which activates the ATR signaling pathway and leads to TIFs. RPA is normally recruited at telomere overhangs during DNA replication, at a time when POT1 is partially released from the telomere, but is replaced by POT1 at the end of DNA replication. Interestingly, transient ATM- and ATR-mediated DNA damage signaling occurs even at normal human telomeres that are completing DNA replication (Cicconi, 2016).

Although 3' overhangs are prevalent among telomeres of organisms with telomerase, in Caenorhabditis elegans 5' overhangs are as abundant as 3' overhangs, and blunt-ended telomeres have been found in Arabidopsis thaliana. 5' overhangs have been also found in mouse and human cells, particularly in G1/S arrested and terminally differentiated cells, as well as in cancer cells that exploit the alternative lengthening of telomeres (ALT) pathway for telomere maintenance (Cicconi, 2016).

In fission yeast, telomeric DNA is protected by a complex that is architecturally reminiscent of shelterin and contains the TRF1 and POT1 homologues Taz1 and SpPot1. In budding yeast, there is not a shelterin complex and the shelterin functions are fulfilled by Rap1 and the RPA-like complex Cdc13-Stn1-Ten1 (CST). Cdc13 does not share homology with POT1, but both proteins use oligonucleotide/oligosaccharide-binding (OB)-fold domains to bind ssDNA. The CST complex exists also in mammals, where it coordinates telomerase-mediated DNA elongation and fill-in synthesis during telomere replication; however, its function is not restricted to telomeres, as it also plays a general role in DNA replication (Cicconi, 2016).

In Drosophila, there is not telomerase and telomeres are elongated by the targeted transposition of three specialized non-LTR retrotransposons (HeT-A, TART and TAHRE). In addition, abundant evidence indicates that Drosophila telomeres can assemble independently of the sequence of the DNA termini. Drosophila telomeres are capped and protected by the terminin complex, which includes HOAP, Moi and Ver. All these proteins interact with each other and share the same features as the shelterin subunits: they are specifically enriched at telomeres throughout the cell cycle and do not perform other functions elsewhere in the genome. Most likely, terminin also includes HipHop, another fast evolving protein that interacts with HOAP and shares the shelterin-like properties of HOAP, Moi and Ver (Cicconi, 2016).

This study focuses on the Verrocchio (Ver) protein, which contains an OB-fold domain with structural similarity to Stn1/RPA2 OB fold. Ver interacts with Modigliani (Moi), and Moi and Ver are both HOAP-dependent and mutually dependent for their telomeric localization. Ver has been also implicated in the recruitment of the HeT-A encoded ORF1p protein and HeT-A transcripts at the telomere. Both electrophoretic mobility shift assay (EMSA) and atomic force microscopy (AFM) showed that Ver binds ssDNA in vitro. Moi was shown not to bind DNA, andVer interaction with Moi was shown to be necessary for Ver localization at telomeres but not for its binding to ssDNA. Finally, it was demonstrated that loss of Ver favors RPA accumulation at telomeres and triggers DNA damage signaling. This suggests that Ver is a functional analog of ssDNA binding proteins such as yeast Cdc13 and human POT1 (Cicconi, 2016).

Previous work has shown that the integrity of the Ver OB-fold domain is dispensable for Ver recruitment at telomeres but is crucial for telomere protection from fusion events. These results suggested but did not prove that Ver possesses ssDNA binding activity. This study provides strong evidence that Ver binds ssDNA. EMSA experiments showed that Ver-GST binds ssDNA probes of different sequence, and that this binding is reduced by competition with ssDNA but not dsDNA. In addition, AFM experiments unambiguously showed that Ver binds DNA with a strong preference for the terminal regions of DNA molecules that end with either 3' or 5' ssDNA overhangs. Collectively, both the results of these experiments and previous studies on Drosophila telomeres strongly suggest that Ver binds ssDNA in a sequence-independent manner. However, it cannot be excluded that diverse DNA sequences could bind Ver with different affinities (Cicconi, 2016).

It was also shown that Ver binds ssDNA as a dimer or a multimer. The protein domain required for Ver-Ver interaction was mapped and it was shown that in the absence of this domain Ver is unable to bind ssDNA and to protect telomeres from fusion events, providing additional evidence that the Ver capping function relies on intact ssDNA binding activity. The presence of ssDNA at Drosophila telomeres has never been directly demonstrated, as the variability of fly telomeric DNA prevented successful application of the commonly used DNA sequence-based methods to characterize the structure of chromosome ends. The findings that Ver binds ssDNA and is required for telomere capping strongly suggests that fly telomeres do in fact terminate with a ssDNA like those of yeasts, plants, and mammals. Studies on C. elegans have shown that this species possesses both 3' and 5' overhangs that are bound by 2 different proteins, CeOB1 and CeOB2, which exhibit specificity for G-rich or C-rich telomeric overhangs, respectively. These data would suggest that Ver could bind both 5' and 3' overhangs. However, they do not prove that these overhangs coexist in living flies (Cicconi, 2016).

The results indicate that Ver binds ssDNA with low affinity, as even high protein concentrations were not sufficient to significantly reduce the amount of unbound probe. However, in a very recent study, Zhang (2016) showed that a trimeric complex formed by recombinant Tea, Moi and Ver, purified with the baculovirus system, has robust sequence independent ssDNA binding activity, while a Moi-capping complexes to maintain an interaction with telomeres (Cicconi, 2016).

Results on Ver provide two important additional pieces of information on the evolution of Drosophila telomeres. First, the findings indicat-Ver subcomplex and ssDNA is probably due to the protein tags and purification methods they used. On the other hand, they clearly showed that Moi, Tea and Ver have high ssDNA binding activity when they act as a trimeric complex. Tea has not obvious ssDNA binding motifs, and remains to be determined whether Tea has its own ssDNA binding activity or simply enhances Ver binding activity (Cicconi, 2016).

The low ssDNA binding affinity of the Ver protein is likely to reflect specific functional requirements. For example, it is conceivable that Ver low affinity for ssDNA prevents unwanted binding of Ver to other ssDNA regions such as those formed during normal DNA replication. It should be noted that telomeric proteins that bind ssDNA with relatively low affinity independently of the sequence have been previously described in yeasts and mammals. For example, Pot1 of S. pombe possesses an N-terminal OB fold that binds DNA in a sequence-dependent fashion, and a C-terminal OB fold with sequence-independent binding properties, a feature that is likely to reflect the need to protect the degenerate telomere sequences present in this yeast species. Another ssDNA binding protein that exhibits no preference for telomeric substrates is C. albicans Cdc13. As a consequence, while S. cerevisiae Cdc13 is recruited at telomeres through sequence-specific interaction with telomeric DNA, recruitment of C. albicans Cdc13 relies on protein-protein interactions. Remarkably, also a high-affinity ssDNA binding complex such as TPP1-POT1 is recruited at telomeres by TIN2, which bridges these ssDNA binding proteins to the dsDNA binding proteins TRF1 and TRF2. Most likely, also Ver recruitment at telomeres depends on interactions with other terminin components and not with telomeric DNA. This is suggested by the behavior of VerΔC. Although this truncated Ver moiety fails to bind ssDNA and to prevent end-to-end fusions, it is normally recruited at telomeres (Cicconi, 2016).

Previous work has shown that Ver and Moi are both mutually dependent and HOAP dependent for their localization at telomeres HOAP binds dsDNA and coats up to 10 kb of telomeric DNA. These findings suggested that HOAP could mediate Ver and Moi recruitment at telomeres. However, recent work has shown that Moi and Ver association with telomeres is also dependent on Tea, which requires HOAP for its telomeric localization. Because HOAP localizes normally at telomeres in tea mutants, these findings suggest that Tea, in the presence of HOAP, could mediate Ver and Moi recruitment at telomeres (Cicconi, 2016).

Although the pathways leading to end-to-end fusion in Drosophila have not been fully elucidated, this study has provided evidence that the early steps of telomere dysfunction recognition are conserved between mammals and flies. This study has shown that fly telomeres depleted of Ver-Moi accumulate RPA and γ-H2AV just as mammalian telomeres lacking TPP1-POT1. It is likely that in the absence of Ver-Moi the telomeric ssDNA binds RPA, which is known to bind ssDNA with high affinity; RPA is then likely to recruit the DNA repair machinery that leads to the formation of telomere associated γ-H2AV foci (Cicconi, 2016).

Several studies in mammalian cells have shown that following POT1 or TPP1-POT1 depletion RPA is recruited at telomeres, leading to the model that loss of POT1 unmasks the single-stranded G overhang, which binds RPA and ATR, eliciting the DDR response. However, it has been recently shown that POT1 is also required for proper telomere replication, probably acting in in the same pathway as CST. These latter findings raise the possibility that RPA localization to POT1-depleted mammalian telomeres is at least in part due to a defect in telomeric DNA replication. The data do not that exclusion of Ver depletion affects telomeric DNA replication in Drosophila. Thus, RPA and γ-H2AV recruitment at ver mutant telomeres could be the consequence of an exposure of the telomeric overhang, a defect in subtelomeric/telomeric DNA replication, or both (Cicconi, 2016).

An interesting issue is how can the ssDNA overhangs of Drosophila telomeres bind Ver in a sequence independent manner and avoid binding by RPA, which has a very strong affinity for ssDNA of any sequence. In human cells, POT1 is less abundant than RPA and, although it specifically recognizes the telomeric DNA sequence, it binds ssDNA with lower affinity than RPA. Nevertheless, after each round of replication, POT1 efficiently replaces RPA at the telomere. The precise mechanism governing this protein switch has not been fully elucidated. It has been proposed that TPP1-POT1 can outcompete RPA when bound to TIN2. An alternative model for the RPA-to-POT1 switch involves TERRA and the heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), which has an RPA displacing activity. It has been suggested that the low TERRA levels during the late S phase favor the hnRNPA1 activity promoting the RPA replacement with POT1. How can Ver replace RPA at the end of DNA replication? This process might be related to dynamic transformations of the Moi-Tea-Ver complex that could modulate its affinity for ssDNA. It is also possible that the physical interaction between RPA and Ver lowers the affinity of RPA for DNA, thus allowing Ver to outcompete RPA for ssDNA binding. However, the precise mechanism governing RPA to Ver switch is currently unknown and will be a goal of future studies. (Cicconi, 2016).

In all organisms studied so far, specialized OB-fold proteins bind telomeric single stranded overhangs ensuring protection of chromosome ends. Past and current findings on Ver broaden the list of these OB fold proteins, and strengthen the concept that the general architecture of telomere complexes is conserved across evolution, despite a remarkable plasticity in the individual components of the complexes. TRF1 and TRF2 shelterin components bind the DNA duplex and are connected to the ssDNA binding protein POT1 by the non-DNA-binding TIN2 and TPP1; the shelterin-like fission yeast capping complex has similar features. It has been suggested that these shelterin complexes are functionally equivalent to the CST and Rap1-Rif1-Rif2 complexes of budding yeast (Cicconi, 2016).

The telomere-capping complexes of yeast, mammals and Drosophila share similar molecular architectures. The human shelterin and the fission yeast shelterin-like complexes have similar architectural features. In both complexes, the proteins that bind the DNA duplex (TRF1-TRF2 and Taz1) are connected to the ssDNA-binding protein POT1 by non-DNA-binding proteins (TIN2-TPP1 and Poz1-Tpz1). Similarly, in Drosophila terminin, HOAP-HipHop, which bind the DNA duplex, are bridged to the ssDNA-binding Ver by Moi, which does not bind DNA. Tea directly binds Ver and Moi but it is currently unknown whether it binds DNA. It has been suggested that the POT1-TIN2-TPP1 and Pot1-Poz1-Tpz1 subcomplexes are functionally equivalent to the CST complex of budding yeast, which binds ssDNA through its Cdc13 subunit, while the Rap1-Rif1-Rif2 complex binds the DNA duplex (see text for detailed explanation and references (Cicconi, 2016).

The finding that Ver but not Moi binds ssDNA suggests that terminin and shelterin have similar molecular architectures. Drosophila HOAP and HipHop interact with each other and are mutually dependent for their stability. In addition, ChIP analysis has shown that the two proteins are enriched over the terminal 10 kb of the chromosomes. Thus, even if HipHop binding to DNA has never been directly demonstrated, it is likely that the HOAP-HipHop subcomplex binds the DNA duplex. Moi binds both HOAP and Ver, and thus is likely to bridge dsDNA-binding HOAP-HipHop with ssDNA-binding Ver. AP/MS experiments have shown that Moi and CG30007 (Tea) are the most abundant Ver-interacting proteins, suggesting a functionally relevant interaction between the three proteins. Tea does not contain any known DNA binding domain and its DNA binding properties have not so far been investigated. Should Tea fail to bind DNA, then the structural similarity between shelterin and terminin would be even greater. In both complexes, there would be a pair of proteins (TRF1-TRF2 and HOAP-HipHop) that bind the DNA duplex, a single ssDNA binding factor (POT1 and Ver) and two non-DNA-binding proteins (TIN2-TPP1 and Moi-Tea) connecting the dsDNA- and ssDNA-binding subcomplexes. Thus, although the shelterin and terminin components do not share any sequence homology, they form multi-protein complexes with similar molecular architectures (Cicconi, 2016).

It has been proposed that concomitant with telomerase loss Drosophila rapidly evolved terminin, a telomere-specific protein complex that binds and protects chromosome ends independently of their DNA sequence. It was also proposed that Drosophila non-terminin telomere-capping proteins correspond to ancestral telomere-associated proteins that could not evolve as rapidly as terminin because of the functional constraints imposed by their involvement in diverse cellular processes. This hypothesis is supported by the fact that the many non-terminin proteins required for telomere capping (HP1a, ATM, Rad50, Mre11 and Nbs) have homologues playing roles at human and yeast telomeres. Additional support for this hypothesis has been provided by recent findings on separase and pendolino/AKTIP. The conserved protease separase has been shown to be required for telomere protection in both Drosophila and humans. Pendolino (peo) prevents telomeric fusions in flies while its human homologue AKTIP is required for telomere replication. Strikingly, Peo and AKTIP directly bind unrelated terminin and shelterin components, indicating that they co-evolved with divergent capping complexes to maintain an interaction with telomeres (Cicconi, 2016).

Results on Ver provide two important additional pieces of information on the evolution of Drosophila telomeres. First, the findings indicate that the terminin proteins (HOAP, HipHop, Moi, Ver and possibly Tea), although fast-evolving and non conserved outside the Drosophilidae family, are likely to form a telomere-capping complex that is architecturally similar to the shelterin complex. Second, this study has shown that Drosophila telomeres are likely to terminate in ssDNA overhangs that recruit RPA just like the yeast and human telomeres. Moreover, like in human telomeres, the levels of telomere-associated RPA and γH2AV (γH2AX) substantially increase when telomeres are depleted of proteins that bind the terminal ssDNA. Collectively, these results reinforce the idea that apart the capping complexes and the mechanisms of telomere length maintenance, Drosophila telomeres are not as different from human telomeres as generally thought. It is thus believed that Drosophila is an excellent model system for studies on telomere organization and function, which can also be exploited for the identification of novel human proteins involved in telomere maintenance (Cicconi, 2016).